This invention relates to a highly effective hypoglycaemic protein called polypeptide-k, extracted from Momordica charantia. This invention also provides a method for the extraction of said polypeptide-k from Momordica charantia. Further, the invention provides a novel hypoglycaemic composition employing the said protein, and useful in the treatment of diabetes mellitus.
Insulin has hitherto been commercially synthesized from the pancreas of animals and human insulin from E. coli (Eli Lily, U.S.A.). So far there is no report of commercial extraction of insulin like polypeptide from plant source.
Isolation of insulin from animal pancreas is open to objection due to the following reasons:
1. By killing 10,000 animals only one pound of pure insulin is obtained.
2. It is not being sublingually administered.
3. If the pancreas is infected by some diseases there is always a probability of its being carried (if it is a virus) along with the insulin.
4. Human insulin can be synthesised from E. coli which is expensive.
Hence, to obviate these and other drawbacks in conventional insulin extraction methods, scientists focussed on plant based products.
Momordica charantia is a perennial herb of the family Cucurbitaceae, widely grown in Asia. The herb is endemic to tropical regions like India, S. Africa, Philippines, China and Burma. The species of Momordica found in western countries are different from the tropical species in that, the plants differ in morphological and organoleptic properties.
Various parts of this plant, especially the fruits, have been widely used for preparation of hypoglycaemic pharmacological compositions.
In Indian Patent No. 136565, the applicant has disclosed a method for the extraction of a protein called xe2x80x98polypeptide-pxe2x80x99 from Momordica charantia. The dried and pulverized fruits and tissue cultures of Momordica are separately extracted in ethanol and then mixed with cold ethanol and diethyl ether. Thereafter, needle-like crystals are formed by adding zinc in traces after 18 hr. The fruits and cultures are separately crushed, homogenized in water, ethanol and concentrated sulfuric acid is added for adjusting pH to 3, thereby obtaining flocculent precipitates.
This method had the following drawbacks:
1. The use of alcohol in the extraction procedure was not practical due to its unavailability in large amount and the impurities present in it.
2. The use of raw material as fruits and tissue culture creates problems in handling, uneconomically viable and the yield was very poor.
The drawbacks of this patent were obviated in another Indian Patent No. 176040. This patent discloses a process for extraction of a highly effective polypeptide-p by using hexane along with diethylether. Although the process developed and disclosed in above referred patent resulted in good yield, improved purity and high efficacy of the drug by removal of oil and sapogenins and other contaminants therefrom, yet, it had a few drawbacks, some of which a few are given below:
1. The purification of polypeptide-p was a cumbersome method due to the presence of interfering radicals as oil and sapogenins.
2. Use of diethylether in the extraction procedure was not practical due to its highly inflammatory nature and high cost.
3. The presence of pesticides/insecticides/urea and other contaminants affected the purity of polypeptide-p.
4. The yield was not optimum.
This protein called xe2x80x98polypeptide-pxe2x80x99 (SEQ ID NO: 1) was extracted from the fruits and tissues of Momoridca charantia. This protein comprised amino acids as shown in Table 1 below:
U.S. Pat. No. 5484889 describes a plant protein useful for treatment of tumors and HIV infection. The protein has been obtained from the seeds of Momordica charantia. It is pertinent to note that this protein isolated and purified is a ribosome inactivating protein and hence, useful in tumor therapy. The processes described for the extraction of the protein involve use of solvents and the tedious process of chromatography, dialysis etc. In the processes described in this patent as well as in Indian patent No. 176040, the yield, purity were low and had several contaminants. Accordingly, to obviate these and other drawbacks, the applicant has isolated a novel protein called xe2x80x98poplypeptide-kxe2x80x99 having hypoglycaemic property from Momordica charantia and has also devised a novel process for extraction of the protein from the same source. The letter xe2x80x98kxe2x80x99 is derived from the term xe2x80x98karelaxe2x80x99 which means xe2x80x98bitter gourdxe2x80x99 or Momordica charantia in a main Indian language.
The main object of the invention to provide a novel protein called xe2x80x98polypeptide-kxe2x80x99.
It is an object of the invention to provide a process for the extraction of a protein called xe2x80x98polypeptide-kxe2x80x99 from the dry seeds of Momordica charantia. 
Another object is to prepare a novel hypoglycaemic composition using the said protein.
Yet another object of the invention is to provide a hypoglycaemic composition containing xe2x80x98polypeptide-kxe2x80x99 for treatment of diabetes in human beings and animals.
Still another object of the invention is to provide a novel protein called polypeptide-k which is capable of reducing high blood pressure and increasing immunity in human beings and animals.
One more object of the invention is to provide a novel protein namely polypeptide-k which takes care of neuropathy and makes the patient feel normal.
In accordance with the above and other objectives, the invention provides a novel protein xe2x80x98polypeptide-kxe2x80x99 extracted from Momordica charantia, a process for the extraction of the said polypeptide-k and a novel hypoglycaemic composition employing the novel polypeptide of the invention.
Accordingly, the invention provides a method for the extraction of proteins from dry seeds of Momordica charantia, said process comprising the steps of:
i) grinding the dry seeds to a fine powder in a suitable mill,
ii) treating the pulverized seeds with a mixture of hexane-acetone,
iii) dissolving the residual mass in about 80% aqueous acetone,
iv) adjusting the pH upto 9.5 by adding suitable organic buffer like ammonium hydroxide,
v) treating the supernatant layer with sulfuric acid,
vi) collecting the flocculent precipitate of polypeptide-k and isolating the protein by selective crystallisation. Thereafter, the protein is analysed by chromatography.
The protein isolated in the present invention i.e. xe2x80x98polypeptide kxe2x80x99 is different as compared to the protein in the prior art. To describe in detail, the protein isolated in the present invention is a protein having 18 amino acids, and is called xe2x80x98polypeptide kxe2x80x99. The process for extraction of the protein consists of de-oiling of the washed seeds of Momordica charantia, using hexane and a little acetone in the ratio of 3:1. The dry seeds are used because polypeptide-k is a storage protein and it gets accumulated in large quantities when the seed is dried. After de-oiling, the seeds are dried, powdered and dissolved in water and acetone taken in the ratio of 3:1. A mixture is made and then the pH is adjusted to 9.5 by adding ammonium hydroxide. Supernatant was remove and the pH was adjusted to 3 by adding sulfuric acid. The flocculent precipitate was collected and dried. The dried mass was powdered and washed with water and acetone to remove oil, salts and other undesirable material, till it gives a single spot in TLC and HPLC.
In one embodiment, the dried seeds of Momordica charantia are split, washed thoroughly with water 2-3 times to render it substantially free from impurities and dried under vacuum, before extraction of the protein.
In another embodiment, the solvents used for removal of the oils from the seeds comprise a mixture of hexane and acetone in the ratio of 3:1.
In one embodiment, thin glass plated (20xc3x9720) coated (0.4 mm to 0.5 mm thick) with silica gel G are activated at 100xc2x0 C. The solution of insulin is applied, the plates developed in n-butanol, acetic acid water (12:5:2) are dried, and single spot nearly corresponding to standard insulin visualized by spraying nin-hydrin (0.25%) in acetone, isolated along with silica gel G from unsprayed plates, extracted in 50% ethanol buffered with ammonium hydroxide or 10% of formic acid, filtered, the filtrate dried and pure white needle-like crystals formed.
In yet another embodiment, when the analysis is carried out, the isolated substance is hydrolyzed along with the standard insulin, applied on paper chromatograms separately, developed, yielding 18 amino acids including glutamine. This isolated substance is a protein named as xe2x80x98polypeptide-kxe2x80x99.
In another feature, the isolated substance and the standard insulin are hydrolyzed separately by 6 N HCI for 20 hours, dried, reconstituted in 50% ethanol, applied on Whatman No. 1 filter paper strips developed in n-butanol, acetic acid, water (60:20:20), strips developed sprayed with 0.25% nin-hydrin in acetone. The standard hydrolyzate shows presence of 18 amino acids including glutamine.
In the analysis is carried out, the seeds are extracted in hexane acetone yielding a product which has a melting point (234xc2x0 C.), Gel electrophoretic pattern of the accompanying drawings and number of amino acids of the standard insulin except glutamine being extra in polypeptide-k.
It may be noted that most of the plant parts of Momordica contain the protein disclosed by the invention, in varying degrees. As such, the protein polypeptide-k may be extracted from the dry seeds.
The dried seeds are processed using hexane (food grade) along with acetone instead of ether as used in the process described in earlier Patent No. 176040. The process has resulted in high yield, improved purity and high efficacy of polypeptide-k by removal of undesired oils, flavonoids and sapogenins therefrom.
The Applicant has analyzed the peptide isolated from Momordica charantia and found that this protein has 18 amino acids. The 18th amino acid is glutamine. The UV spectrum has shown absorption peak at 275 nm whereas the peak for polypeptide-p, i.e. another protein found in Momordica charantia is noticed at 250 nm. BPLC analyses for the protein polypeptide-k shows a single peak. Thus, the mass spectrum analyses done for polypeptide-k discloses the fact that polypeptide-k consists of two peptide chains as opposed to a single chain present in polypeptide-p.
Another notable feature is that polypeptide-p can be isolated from the fruits, fresh seeds and tissues of Momordica charantia. Whereas, polypeptide-k is obtained from the dried seeds of Momordica charantia as a storage protein.
More importantly, the earlier isolated polypeptide-p has approximate molecular weight of 11,000 kd whereas polypeptide-k of the present invention has an approximate molecular weight of 18,000 kd.
Thus, polypeptide-k (SEQ ID NO:2) differs from polypeptide-p in the following respects:
1. The polypeptide-k has 18 amino acids whereas polypeptide-p has only 17 amino acids. The extra amino acid present in polypeptide-k is glutamine.
2. The approximate molecular weight of polypeptide-k is 18,000 units whereas the weight of polypeptide-p is 11,000.
3. Polypeptide-k has a free N-terminal.
4. Polypeptide-k is not water soluble whereas polypeptide-p is partially water soluble.
5. Polypeptide-k is not injectible to a patient and can be administered orally only through sub-lingual route whereas polypeptide-p is injectible as described. In other words, polypeptide-p is injectible (intramuscular) and this is inconvenient to patients, however, polypeptide-k is taken sublingually from the above surface of the tongue and its administration and absorption is easy, acceptable and convenient to patients.
6. Polypeptide-k is stable and the life is about 18 months (kept at normal pressure and temperature). On the other hand, polypeptide-p is unstable and its life is hardly 2-3 months when kept at normal pressure and temperature.
7. Polypeptide-k has the combustion point (m.p) of 234xc2x0 C., whereas polypeptide-p has early combustion point which starts at 228-232xc2x0 C.
Diabetes is a disease wherein glucose is not utilized as an energy source in the body such glucose remains at a high levels in the blood and eventually gets excreted through urine. In some conditions, insulin secreted from beta cells of pancreas is insufficient or does not sufficiently fulfill its function.
Diabetes is generally classified into insulin-dependent diabetes (Type I diabetes) and non-insulin-dependent diabetics (Type II diabetes). Type I diabetes is in the state of lowering of the function of pancreatic beta cells resulting from hereditary cause, viral infection obesity, drug effect, accident, etc. wherein insulin is not efficiently secreted, and suddenly attacks mainly in the twenties to thirties. Although it is not sure, onset of type II diabetes in the forties or in cases with family history of diabetes, obesity, stress, etc. In the case of type II diabetes, since insulin is sufficiently secreted from pancreas but insulin resistance and glucose utilization are different from those of normal person, blood sugar is not returned to normal level in spite of hyperinsulinemia.
Diabetes is accompanied with numerous symptoms. Typical examples of such symptoms are polyuria, excessive drinking and polyphagia. That is, diabetic patients exhibit polyuria which is caused by excretion of glucose and excessive water through urine by the action of osmotic pressure originated from high blood glucose level, and therefore, complain of thirst caused by dehydration, which induces excessive drinking, and causes the empty of stomach to intake excess of food. Diabetic patients cannot efficiently utilize glucose as an energy source and, instead, utilize protein and fat as preserved in the body, and this phenomenon is caught in a vicious cycle causing reduction in body weight.
However, such phenomena are merely acute symptoms observed in the primary stage of diabetes. If diabetes becomes chronic by delay of treatment, chronic vascular diseases add up as complications. Thus, diabetic complications such as diabetic retinopathy (visual disturbance, blindness, retinal hemorrhage), diabetic nephropathy, diabetic peripheral neuropathy, etc. reduce general metabolic and sensory function of human body.
A number of medicines have been produced and tested act to lower blood sugar of a non-insulin dependent diabetics. However, majority of these medicines have one or more undesirable features, some of them have significant side effects for a large portion of the population, or a large dosage is necessary. Also, some of them reduce the blood sugar level too much so that they can only be used sporadically or they can be a threat to health, and others have possible toxicity. At present, there is no natural antidiabetic drug which is highly effective at lowering blood sugar, yet does not lower it to an unsafe level, and has no significant side effects.
According to the present invention, a pharmacologically active hypoglycaemic agent is produced in a simple and straightforward way using only the protein of the invention.
Since it is very effective, relatively small amounts of the homeopathic medicine need be ingested in order to reduce the blood sugar level.
The protein extracted from Momordica charantia exhibits hypoglyceamic properties and accordingly compositions comprising the protein can be used for the treatment of hypoglycemia in mammals. The protein obtained from Momordica is in the form of an amorphous powder. The protein activates the inactive insulin and, thus, it can rejuvenate the pancreas depending upon the chronicity of the pathological condition of the individual. In fact, in course of time, it may act as a cure for diabetes. The applicant has conducted more than 500 experiments and confirmed that the single dose to about 12 mg to 70 mg of the protein at a time is quite effective. Accordingly, it is advisable that compositions containing the protein in single dose should comprise about 12 mg or more of the protein.
Hypoglyceamic compositions using the proteins of the invention can be formulated in a variety of physical forms such as tablets, edible products. For preparation of a tablet, about 12 mg to 70 mg of the protein is mixed with pharmacologically acceptable carriers suitable for consumption. The pharmacologically acceptable carrier must be of sufficient purity non-toxicity and should not interfere with the activity/efficacy of polypeptide-k. Edible products like biscuits, chewing gums, losenzes etc., which are not instantly swallowed can be prepared. In all such preparations, the content of the protein is about 12 mg to 70 mg. It is found that low salt biscuits prepared using the protein of the invention are very popular with diabetics.
It is pertinent to note that the hypoglyceamic composition of the invention is to be consumed 10 minutes before meals, at least 4 times a day. The most important aspect is that the tablet or the hypoglyceamic composition should only be chewed and should not be swallowed instantaneously.
In a feature of the invention, the hypoglyceamic composition herein described has no side effects. It can be consumed without restricting the use of other therapies. It has no cross reaction with insulin.